FVHO acts on fructosyl valyl histidine in the presence of oxygen, and catalyzes a reaction for producing glucosone, valyl histidine, and hydrogen peroxide. Together with protease, FVHO has been conventionally used as a crude enzyme for a hemoglobin A1c measurement reagent, and this enzyme alone has been used for an enzyme sensor. The hemoglobin A1c value in the blood is an indicator of long-term glycemic control. As the source of the FVHO, the genus Coniochaeta, genus Eupenicillium (Patent Literature 1), genus Phaeosphaeria (Patent Literature 2 and Non-patent Literature 1), genus Emericella (Patent Literature 3), etc., have been known.
In general, crude enzymes used for reagents or sensors take various preparation forms such as a solution state and a dry state according to the purpose. For example, when used for a hemoglobin A1c measurement reagent, FVHO is often stored in the form present in a solution, and when used for a hemoglobin A1c sensor, FVHO is often stored in the dry state. Further, many of the enzymes used for reagents or sensors are distributed as products in the dry state. (Hereinbelow, preparations in the solution state are sometimes referred to as solution preparations or liquid reagents, and preparations in the dry state are sometimes referred to as dried preparations.)
Products in the dry state (dried preparations) are easy to handle in preservation and transportation because of their lightness and small volume, as well as reduced concern for spoilage by microbial contamination because they are dried. Moreover, products in the dry state can be developed for various applications because the enzyme dissolution concentration can be freely adjusted according to the usage, and the kind of buffer for dissolution can be selected as desired. Further, in general, products in the dry state can stably maintain enzyme activity for a long period of time compared to products in the solution state.
In contrast, preparations in the solution state (solution preparations) do not need to be dissolved at the time of use; therefore, operation is simple, which advantageously reduces the probability of contamination and mistaking one solution for another.
For liquid reagents, even when the stability is poor, correct results can be obtained as long as calibration is performed in each measurement using a reference material; however, since calibration is not performed in sensors, further improvement in storage stability is required.
In any form of use, addition of a stabilizing agent to protect an enzyme protein and prevent modified deactivation is essential. A stabilizing agent to be added to an enzyme product requires not only the ability during formation into products to prevent modified deactivation of an enzyme protein due to drying but also the ability to prevent activity loss during storage or distribution process.
To produce a dried preparation, various means can be used to make an enzyme in the dry state. Examples include a method in which an object enzyme is precipitated from a solution containing an enzyme protein using an organic solvent such as acetone and alcohol, thus collecting the object enzyme to form a dry powder; a spray-dry method in which a solution containing an enzyme is nebulized, followed by drying with hot air; and a freeze-drying method in which a solution containing an enzyme is frozen, followed by drying under reduced pressure.
When an enzyme is dried, problems such as activity loss due to protein modification and generation of turbidity matter during redissolution may occur. In many such cases, a stabilizing agent to protect an enzyme protein and prevent modified deactivation is added.
A known method for stabilizing a freeze-dried preparation of an enzyme having FVHO activity is the technique of allowing ethylenediaminetetraacetic acid and sodium glutamate, or calcium chloride and trehalose to coexist with the enzyme to freeze-dry the enzyme (refer to Patent Literature 4).
A known method for stabilizing an FVHO solution preparation is the technique of adding ethylenediaminetetraacetic acid and allowing at least one member selected from the group consisting of ammonium sulfate, xylitol, and glycine to coexist with FVHO to freeze-dry the FVHO (refer to Patent Literature 5).